Treatment of blood, blood cells, plasma, serum, plasma fractions, other biological fluids and protein solutions by irradiation has been a widely investigated approach to inactivate pathogenic viruses, in particular, small single-stranded non-enveloped DNA viruses. Examples of irradiation treatment currently under study include short-wave ultraviolet light, long-wave ultraviolet light, visible light with photosensitizing compounds, and broad-spectrum high-intensity flash light.
In the 1930's, small volumes of autologous whole blood were exposed to UV irradiation from a medium-pressure mercury vapor lamp to inactivate infectious organisms. By re-infusion of the irradiated blood, an apparent immunostimulatory effect was observed.
Between 1946 and 1955, treatment of pooled plasma with UV-C light to inactivate infectious agents had been investigated in the United States. The first encouraging results lead to UV-C treatment at 253.7 nm as a minimum requirement for therapeutically applicable human plasma (Murray et al. 1955). With the introduction of electronic spectrophotometry, plasma as well as virus culture solutions containing plasma or serum were determined to be optically opaque to 253.7 nm-light with a penetration depth of less than 1 mm (Suhrmann and Kollath 1928). Therefore, various thin-film irradiators were constructed for large-volume plasma sterilization or vaccine attenuation. Examples include the Habel-Sockrider irradiator (Habel and Sockrider 1947), the Milzer-Oppenheimer-Levinson centrifugal film generating device with water-cooled lamps (Milzer et al. 1945; Benesi 1956; Taylor et al. 1957a; Taylor et al. 1957b; McLean and Taylor 1958; Oppenheimer et al. 1959), and the Dill irradiator (Murray et al. 1955) (Dill Instruments Co.), which is the only design still commercially available.
Despite the foregoing advances, serum hepatitis persisted (Neefe 1949; Barnett et al. 1950; James et al. 1950). Exhaustive studies determined that UV doses required to inactivate Hepatitis B virus severely reduced biological activity of plasma proteins, and that method was abandoned in the late 1950's (Murray et al. 1955; Kallenbach et al. 1989). In 1958, an improved combination administration of beta-propiolactone and subsequent UV-C irradiation was introduced. This process was able to inactivate serum hepatitis viruses but leave intact at least those biological activities of prothrombin complex, immunoglobulins, and albumin (Smolens and Stokes 1954; Hartman et al. 1955; Prince et al. 1983). From 1968 onwards, this process was applied by commercial plasma fractionators in manufacturing of combined albumin-immunoglobulin serum concentrate, and from 1976 onwards, of a prothrombin complex concentrate with use of a Dill thin-film UV irradiator (Stephan et al. 1981, Stephan 1982a, 1982b). Infectivity studies in chimpanzees and later in cell cultures showed an effective inactivation of hepatitis A, B and C (Heinrich et al. 1982, 1987, Frösner et al. 1983, Prince et al. 1983, 1984, Stephan 1989), while HIV proved to be more resistant against UV-C (Dichtelmüller et al. 1987, 1993). The apparent insufficiency of the beta-propiolactone/UV-C treatment to inactivate the AIDS virus HIV in a prothrombin complex concentrate (Kleim et al. 1990, Kupfer et al. 1995) and the availability of other physical and physicochemical inactivation methods, such as heat or detergent treatment, led to the abandonment of the beta-propiolactone-UV-C method in 1990 (Pustoslemsek et al. 1993).
In the 1980's, Dichtelmüller et al. (1987) and Kallenbach et al. (1989) confirmed that UV-C alone would require excessively high doses to inactivate HIV in blood plasma, and UV-B was ineffective for pathogen inactivation (Prodouz et al. 1987).
On the other hand, small non-enveloped DNA viruses that are insufficiently susceptible to physical and physicochemical inactivation methods, are effectively inactivated by UVC light. A paradigm is the family of parvoviruses, such as Kilham Rat Virus (Proctor et al. 1972), Murine Minute Virus (MMV) (Harris et al. 1974, Rommelaere et al. 1981), and Porcine Parvovirus (PPV) (Brown 1981). For example, U.S. Pat. No. 6,190,608 describes a UV-C 253.7 nm fluence (the irradiant energy incident at the sample distance divided per area, expressed as erg/mm2, mJ/cm2 or J/m2) of 12 mJ/cm2 inactivated MMV in a solution containing 2 mg immunoglobulins/mL.
In addition, a fluence of 9-25 mJ/cm2 is sufficient to inactivate parvoviruses in a concentrated solution of fibrinogen (WO 96/02571) or a dilute solution of purified coagulation factors (JP patent publ. 196531/1995). Higher UV dose of 100 mJ/cm2 have been reported for the necessary minimum for plasma (Chin et al. 1995), fibrinogen (Marx et al, 1996), albumin, immunoglobulins (Hart et al. 1993; Chin et al. 1997), and animal sera (Kurth et al. 1999), although fluences as low as 50 mJ/cm2 effectively inactivate parvoviruses (Chin et al. 1997).
Other examples for treating biological samples, such as plasma and red blood cells, include generation of singlet oxygen by a photosensitizer and light, or by flash-photolysis effects of high-intensity, broad-spectrum light flashes from a Xe tube. The technical advances in electronic light measurement and the availability of digital radiometers facilitated determination of irradiance from a light source, so that fluence data are routinely determined and disclosed.
Plasma proteins have demonstrated a high sensitivity to UV or photosensitized irradiation. The alteration of UV-C-irradiated plasma and plasma proteins resulted in prolonged coagulation time (Cutler et al. 1950; Cutler et al. 1955), changed electrophoretic mobility (Hellbrügge and Marx 1952; Larin 1958), aggregation (Engelhard and Eikenberg 1955), alteration of sedimentation properties in the ultracentrifuge (Claesson 1956), and antibody titer reduction (Battisto et al. 1953; Kleczowski 1954). For example, UV-C treated fibrinogen shows decreased clot elasticity (Di Benedetto et al. 1963a, 1963b, 1963c) and delayed clotting at >100 mJ/cm2 (Marx et al. 1996); factor VIII activity is reduced in UV-C treated plasma (Kallenbach et al. 1989; Chin et al. 1995); fibrinogen clotting time is markedly increased, and von Willebrand factor and factor VIII activity is slightly decreased in methylene-blue/light treated fresh frozen plasma (Aznar et al. 1999).
To minimize the deleterious effect of ionizing and UVC radiation on proteins, quenchers of radical oxygen species have been used, in particular the flavonoid rutin (Erdmann 1956, WO94/28210), ascorbic acid (Erdmann 1956), and creatinine (JP 11286453-A) as an UV-C absorbing additive. However, if these additives supplement an additional absorbance at the wavelength used, too little UV-C energy may actually reach the pathogens. However, an excess of applied energy readily damages the proteins, and it is therefore necessary to not exceed the UV or visible light dose required for sufficient pathogen inactivation, and to calculate or determine this dose as accurately as possible.
Another possibility to protect proteins from singlet oxygen, which is generated from dissolved atmospheric oxygen by photoenergetically excited substances such as tryptophan derivatives present in the solution, is to remove the oxygen dissolved in the solution before the irradiation, and to replace the oxygen by an inert gas atmosphere, e.g. by nitrogen flushing, during the irradiation (Henzler and Kaiser 1998).
In relation to the above, electronic radiometers and spectroradiometers are used to measure radiant energy from a light source (J/m2, mJ/cm2) and radiance (W/m2, mW/cm2), or the incident light energy or power termed irradiant energy and irradiance. Such radiometer sensors are usually made of photovoltaic or photoelectric materials, such as semiconductor photocells or photodiodes optionally coated with a luminescence-doped phosphor layer to convert ultraviolet into visible radiation (Latarjet et al. 1953). One main application of radiometer sensors is to monitor light sources. Radiometer sensors have found widespread use in water disinfection and in phototherapy. The spatial response of such sensors is critical for an accurate irradiant power determination e.g. in UV-A and UVB therapy cabinets (Pye and Martin 2000, Martin and Pye 2000). To apply the accurate light dose, these devices have to be validated and calibrated by radiometry (Taylor et al. 2002), but the problem of the intensity decline by lamp aging has been settled only recently by the use of a radiometric target sum dose which has to be attained (Allen and Diffey 2002). A similar approach to compensate protein irradiation experiments for variations in lamp intensity was the use of a radiometric lamp monitor with an integrating counter (Rideal and Roberts 1951).
Electronic sensors mounted at the end of the light path (Taylor et al. 1941) are only of limited use in the irradiation of absorbing biological fluids, and the use of radiometer sensor measurements or signals to determine a or calculate a fluence (dose) or fluence rate (dose rate) distribution in a photochemical or pathogen photoinactivation reactor is limited by the laws of optics. A point source such as a bulb obeys the inverse square law in all directions, while a tubular source such as a fluorescent tube does not. It would in theory be possible to calculate a light energy distribution in an irradiated target only if specific properties were known: reflecting and scattering properties of surfaces; refractive properties of materials; absorption properties of passed and irradiated media. However, models have only been established for very simple systems, such as tubular cells or the elliptical photoreactor (Alfano et al. 1986a, 1986b).
Chemical actinometry measures the effect of light on a photochemical reactant mixture (Kuhn et al. 1989; Favaro 1998). In general, established photochemical actinometers with known quantum yield and temperature dependence can surpass electronic devices in reproducibility and stability. Photochemical reaction products should be measured on-line most conveniently e.g. by spectrophotometry or chemical sensors (Gauglitz 1983). The photochemical approach has therefore found general acceptance to determine both a light source's fluence rate or irradiant power.
A preferred approach employs total opacity of the actinometric solution at the measured wavelength, so that the photochemical reaction occurs only at the very surface (Kuhn et al. 1989; Favaro 1998). Therefore sufficiently high reactant concentrations are preferred.
For UV-C measurement, the classical actinometers are uranyl oxalate (Bowen 1949, Kuhn et al. 1989) and the ferrioxalate actinometer, but use of the first is limited by the Uranium radiotoxicity, and of the latter by UV-B, UV-A, and visible light sensitivity (Kirk and Namasivayam, 1983). Hydrogen sulfite or hydrogen cyanide adducts of triphenylmethane dyes also are as well known as UV-C-sensitive actinometers. For example, the colorless malachite green leucocyanide dissolved in ethanol does not show a long-wave UV or visible light sensitivity, but the green photoproduct absorbs additionally in the UV-C range (Calvert and Rechen 1952, Fisher et al. 1967). Azobenzene (Actinochrome 2R 245/440) in methanol enables a reuse of the actinometric solution (Gauglitz and Hubig 1981, 1984, 1985) as well as heterocoerdianthrone endoperoxide (Actinochrome 1R 248/334) (Brauer and Schmidt 1983). Despite the elegance of these complex organic compounds dissolved in organic solvents, it has been postulated that actinometric substances and solutions should be non-toxic and non-hazardous.
Immediately after various ultraviolet lamps became available in the beginning of the 20th century, acidic iodide solutions have been used as chemical actinometers (Bering and Meyer 1912). The low quantum yield of 0.05 lead to the use of nitrous oxide (N2O) as an electron scavenger (Dainton and Sills 1960, Rahn 1993). A more recent approach of opaque UVC actinometry is the iodate-stabilized photodecomposition of iodide (Rahn 1997, Rahn et al. 1999, 2003). The triodide formed from iodide photolysis at 253.7 nm is determined spectrophotometrically at 352 nm or higher wavelengths (375 nm, 400 nm). This system has the advantage of insensitivity to wavelengths over 300 nm.
The use of an actinometer solution has been proposed to replace UV-sensors by flow-through or static probes containing the actinometer solution, which are inserted into the irradiation reactor. The concentrated actinometer solution, e.g. the aforementioned iodide/iodate actinometer (U.S. Pat. No. 6,596,542) or an uridine solution (Schulz et al. 2001) is pumped through a UV-transparent tube receiving the UV light from the lamp, or contained in a cell with a transparent window facing the UV lamp to be exposed for a defined time, and the photoproducts are then measured in a spectrophotometer. These sensors, however, measure only the fraction of radiation incident on them, but not the average fluence (light dose) effective on the fluid to be irradiated while contained within the irradiation reactor.
In a rather limited number of cases, chemical actinometry has also been a method of choice to determine the number of totally absorbed photons or the effective light dose distribution in photochemical reactors and flash photolysis cells. In the pioneering phase of protein photochemistry in the 1950s, stirred batch cells were more frequently used for protein denaturation than for virus inactivation experiments (Rideal and Roberts 1951, Claesson 1956, Kleczkowski and Gold 1962), and uranyl oxalate actinometry was employed as standard method to determine the total incident quanta. Heidt and Boyles (1951) tested the effect of temperature and current on low-pressure mercury vapor-lamp UV-C-intensity for a 12 mL batch photoreactor using the uranyl oxalate actinometer. Engelhard and Eikenberg (1955) constructed a 50 mL batch recirculation cell for protein denaturation experiments and determined the absorbed UV photons by the chloroacetic acid actinometer. Taylor et al. (1957) and Oppenheimer et al. (1959) described the validation of the Centrifilmer irradiator by both off-line radiometry for lamp tests before use with an electronic tantalum photo-cell and absorbance-matching actinometry, based on quantum yield calculations, not on direct calibration, with uranyl oxalate. Apparently, no definitive results for the effective dose were obtained from this validation approach, as remarked upon the experimental treatment of liquid egg white in the Centrifilmer irradiator for the UV-C inactivation of Salmonella spp. (Ijichi et al. 1964), while at least the surface irradiant power could be determined by the use of radiochromic paper dosimetry (Launer and Hammerle 1964). Alfano et al. (1986a, 1986b), tried to confirm calculations for photochemical reactor modeling by chemical actinometry. Yokota and Suzuki (1995) tested an immersion-well photoreactor configured with multiple lamps by standard ferrioxalate actinometry. Recently, Vincze et al. (1999) noted that such an experimental verification procedure is still not common standard. von Sonntag (1999) described laser flash photolysis experiments, wherein a limited penetration depth of the incident light will result in a spatially uneven distribution of reaction products, nevertheless modeling calculations were applied for a simple rectangular photolysis cell, and to avoid absorption-matching actinometric procedures.
The disinfection of drinking water by UV-C light (Gelzhäuser 1985) has necessitated an actinometric validation of flow-through illuminators to ensure complete sterilization. In general, drinking water has a very low UV-C absorption as long as trace impurities such as Fe(III) or humic acids are absent. Therefore chemical actinometers based on decomposition of hydrogen peroxide (Kryschi et al. 1988) or tert-butanol sensitized potassium peroxodisdulfate (Mark et al. 1990) have been developed. Another UVC sensitive actinometer is based on UV-induced hydroxylation of benzoate in alkaline solution to fluorescent dihydroxy benzoate (Moroson and Gregoriades 1964).
The use of a water-soluble triphenyl methane dye (4,4′,4″-tris-di-β-hydroxyethylaminotriphenyl acetonitrile) as an added actinometer substance is described for the “cold-sterilization” of pathogens in fruit ciders, juices and plant saps by flow-through UV-C irradiation (Koutchma and Adhikari 2002, Adhikari et al. 2002). Equipment for this process is commercially available, e.g. the “Cidersure” or the “Sap Steady” thin-film irradiators manufactured by FPE Inc. of Macedon, N.Y., or the “Light Processed System” coiled tube irradiator manufactured by Salcor Inc., Falibrook, Calif. Ciders and juices obtained from squeezed fruits show high turbidity from suspended particles, and high UV-C absorption from dissolved phenolic compounds and from ascorbic acid, and also a viscosity similar to protein solutions. Clarified apple juice has an absorption coefficient of 9/cm at 253.7 nm. The actinometer substance is added to the highly absorbing juice and irradiated in a collimated-beam apparatus, as used for the absolute UV inactivation kinetics determination of microorganisms in non-absorbing suspension (Bolton and Linden 2003). The absorbance of the actinometer photoproduct at 600 nm increases, but in fact the added actinometer substance will only receive the light quanta fraction corresponding to its absorbance fraction of the total absorbance. The effective dose is then calculated from the destruction of the added actinometer dye. As it can be deduced from an “absorbed dose” of 190 mJ/cm2 inactivating no more than 3 log10 colony-forming units (cfu) E. coli K12/mL in apple juice in a petri dish, the addition of an actinometer substance to the sample itself delivers obviously false results for the effective dose. In non-absorbing suspension, 5 log10 cfu/mL E. coli are inactivated at ˜10 mJ/cm2 (Wright and Sakamoto 1999). However, the dose effective in the solution must be the same as the dose effective on the microorganism. The proposed addition of an actinometer substance to an already absorbing medium is therefore not capable to accomplish an exact effective dose measurement.
A direct actinometric method to ensure the application of the correct amount of UV light in the virucidal flow-through UV-C treatment of protein solutions is based on their UV-C-induced UV-B absorbance increase (WO 03/007998). This absorbance increase is said to correlate with the inactivation of bacteriophage Phi-X 174, which is used as a biological dose indicator. A reverse-calibration of the absorbance increase to the corresponding bacteriophage titer reduction and even to the reduction equivalent dose (RED), can be done based on the obtained data, if a calibration plot using the dose-dependent titer reduction of bacteriophage Phi-X 174 has been established. However, the reduction equivalent dose (RED), which is the standard parameter determined in water UV-C disinfection, is not the same as the average dose effective in the fluid. The reduction-equivalent dose (RED) actually expresses a dose distribution which depends in the efficiency of mixing. Due to the decadic-logarithmic titer reduction of the bacteriophage, underirradiated volume fractions contribute more to the average residual bacteriophage titer than to the actinometric magnitude of the average effective dose, which usually shows an ideally linear concentration-dependent change by the converted light photons.
The biological dosimetry using a photoinactivatable microorganism was the first method to determine the applied dose in the irradiation of plasma (Oliphant and Hollaender 1946), although details on the dosimetry using Aerobacter aerogenes (outdated name for Klebsiella pneumoniae) were not given. During use of UVC for the inactivation of serum hepatitis virus, this bacterial strain was used to validate thin-film irradiators (McCall et al. 1957). In addition, single-stranded DNA bacteriophages of the microviridae family such as S13 (Latarjet and Wahl 1945) and Phi-X (phi chi) 174 (Setlow and Boyce 1960) give a linear decrease of titer with an increasing UV irradiation dose. Biodosimetry based on the inactivation of bacteriophages (e.g. Phi-X 174 (Battigelli et al. 1993) or the single-stranded RNA bacteriophage MS2 (Havelaar et al. 1991)), or the inactivation of Bacillus subtilis spores, has been developed for testing flow-through ultraviolet water disinfectors (Sommer et al. 2001). An absorption coefficient of water can be adjusted with dissolved sodium thiosulphate so that the coefficient reduces UV transmissions (Cabaj et al. 1996). Nevertheless, an electronic radiometer at the end of this light path and efficient flow-through mixing to narrow dose distributions (Cabaj and Sommer 2000) is still required for this method, and the incubation time for spore cultivation does not allow for rapid validation.
The validation of flow-through irradiators for ultraviolet irradiation in a combined beta-propiolactone and ultraviolet “cold sterilization” of plasma proteins also has used bacteriophages (Dichtelmüller and Stephan 1988). However, these processes are limited by fast-flowing fractions with lower and potentially insufficient doses (Qualls and Johnson 1983).
Only liquid layers very thin in relation to the light absorption don't require a fluence rate correction for self-absorption. Otherwise, the decline in fluence with the distance has to be calculated (Morowitz 1950). The effects of light absorption of virus suspensions on UV-C-inactivation kinetics was investigated in pioneer experiments in a side-illuminated 1.5 cm-diameter quartz cell (Taylor et al. 1941). The cell was stirred slowly, and a deviation of the inactivation kinetics, linear at a concentration of 10 μg virus/mL, was observed already at 20 μg/mL and at 200 μg/mL. Using stirred top-illuminated petri dishes, it was demonstrated that shielding of viruses in stagnant deeper layers, into which the light could not penetrate at its initial intensity, was avoided when a virus suspension was stirred, so that the virus inactivation kinetics remained linear (Budowsky et al. 1981).
Parameters of the latest designs of whole blood irradiators have been validated by Ternovoy et al. (1988). Actinometry using water-soluble diazonium salts was chosen to compare efficiencies of thin-film cells or capillary devices. The apparatus described did not exceed an internal diameter of 3 mm and are constructed to expose a maximum internal surface to the UV light. Both high protein concentrations and cellular components absorb incident light at the surface, and laminar flow profiles in the cells or cylinders makes dose distribution more uneven, exposing slow-flowing outer layers to higher UV irradiances compared to faster-flowing inner volumes. Therefore such devices are unsuitable in the context of pathogen inactivation of biological substances.
Most blood plasma irradiation devices are based on a thin-film principle, which limits sample throughput. However, high-throughput irradiators have been developed with liquid layer thicknesses exceeding those UV-C penetration depths seen in the past.
For example, a thin-film irradiator manufactured by Dill Instruments Co. is a widely used instrument for irradiation of biological fluids. In particular, U.S. Pat. No. 5,567,616 describes an inclined, externally illuminated and UV-translucent rotating cylinder enabling biological fluid to flow by gravity downward along an inner wall. Two light sensors, mounted at the upper end on an outer surface and in an inner cavity, measure light intensity transmitted by a fluid film and the film thickness. The Dill irradiator has been used for the beta-propiolactone/UV process and for irradiation of fibrinogen preparations stabilized with cystine (McCall et al. 1957) or rutin (Marx et al. 1997).
U.S. Pat. No. 5,133,932 discloses an UV-C irradiation method for pathogen inactivation in a baffled vessel rotating horizontally around its axis, in which the biological fluid is contained in a lower reservoir and dispersed to form a thin film on the inner wall upon rotation, which is then re-mixed with the reservoir. This principle of a thin-film mixed-batch irradiator has already been applied by Oliphant and Hollaender (1946). In the disclosed invention, the UV-C lamps are inserted into the vessel's neck and mounted parallel to the rotation axis. Only a small volume fraction of the vessel can be filled with the fluid, thus reducing the capacity and the scalability of the method.
U.S. Pat. No. 6,190,608 describes a flow-through method to treat blood products with UV-C radiation to inactivate erythrovirus B19, because, as described above, parvoviruses are susceptible to low doses of UV radiation. In a straight flow-through quartz or UV-C-transparent plastics tube, a turbulent flow can be generated by obstacles or nitrogen bubbles. The UV intensity is adjusted by means of a filter between a lamp and the quartz tube, and UV irradiance is measured by an electronic radiometer. The UV dose is adjusted by setting the flow rate for an appropriate exposure time.
U.S. Pat. No. 6,540,967 discloses an inclined, UV-translucent, and internally illuminated rotating cylinder with the liquid flowing downward by gravity on the outer surface. The film-thickness of the liquid layer is controlled by an interferometer. This apparatus is proposed for inactivation of viruses and mycoplasms in biological fluids.
U.S. Pat. No. 6,586,172 discloses a UV-C transparent flow-through cell wherein a static axial mixer provides sufficient mixing to ensure an equal irradiation of the sample. A method of validation also is disclosed, which uses daylight-insensitive aqueous iodide solutions for monitoring of apparatus irradiance from UV lamps by spectrophotometry of formed tri-iodide at 352 nm (Hackradt 1920, 1922, Rahn 1993). In the examples given in the corresponding WO 00/20045, a 1% NaI solution is used for all products regardless of their UV-C absorption coefficient, such as 4.5% albumin solution, plasma, or concentrated immunoglobulin solution. Neither absorbance nor viscosity of the protein solutions are matched and failure of actinometric lamp monitoring in determination of applied UV dose or equivalent dose effective in the protein solution is therefore evident by use of empirical constants in the dose/inactivation equation. In this connection, to ensure an effective pathogen inactivation with a static mixing apparatus, model calculation parameters such as flow rate, device length, and residence time have to be optimized experimentally.
In general, a known disadvantage of using iodide solutions is low quantum yield of photochemical reaction and nonlinear dose-response (Rahn 1997). For on-line processes, radiometers would enable a time-resolved UV intensity measurement as an indicator of lamp ageing and short-time radiance changes.
WO 01/74407 discloses a portable flow-through device for inactivation of pathogens especially in single donations of blood or blood plasma by UV-C irradiation. This device can be validated using a 1% NaI in 20 mM Tris buffer actinometer, however, this the high absorbance of this actinometer is used to determine an “absolute dose” expressed as energy applied per volume (J/m3). This, however, does not conform to the photochemical concept of fluence as the energy incident on and only partially absorbed by an area (Bolton 1999). Historically, all such experiments of virus inactivation have been performed in buffer suspensions spread in thin films over an area to express the fluence in erg/mm2, mJ/cm2 or J/m2, so that comparison of various methodologies would require fluence data.
US patent application 2003/0049809 A1 discloses a method for the flow-through UV-C inactivation of microorganisms in a fluid, where a secondary flow is superimposed in the helically coiled tube wound around the UV-C lamp. The example of parvovirus inactivation in α1-proteinase inhibitor solution at different protein concentrations has been given, as well as the evaluation of the reactor by the use of the iodide/iodate actinometer solution (Rahn 1993, 1997). Both the titer reduction at a pre-defined UV fluence, which decreases at higher protein concentrations, and the term “light yield” given for the actinometric test result demonstrate that the accurate dose effective in the protein solutions can't been determined by the use of a concentrated actinometer solution.
U.S. Pat. No. 6,329,136 discloses a method for pathogen inactivation in biological fluids using a 20 ns-laser pulses at 248 nm. The experimental setup consists of a KrF excimer laser with its beam targeted at a cylindrical 1 cm quartz cell containing the stirred sample solution. The energy applied is measured by a electronic sensor and expressed as the fluence (mJ/cm2). The fluences necessary for inactivation of viruses in buffer and protein-containing solutions are remarkably different. As it is stated in the description: “The pulsed UV dose at (248 nm) required to eliminate [Blue Tongue virus] BTV from fetal bovine serum, as compared to [phosphate-buffered saline] PBS, is 15 times greater. This difference is perhaps due to the optical properties of the host media (i.e. optical transparency). In PBS, the buffered saline media is an optically clear aqueous solution, whereas fetal bovine serum is a less transparent, complex colored solution which contains UV absorbent chemicals, such as proteins, which do absorb some of the 248 nm UV light energy”, it is apparent that high UV-C absorption of matrix proteins reduces sample volumes effectively illuminated by the incident UV laser light.
U.S. Pat. No. 6,576,201 discloses a cylindrical UV-C transparent flow-through cell with a static outer transparent wall and a turning inner cylinder. In a liquid layer in between, the liquid pumped through the cell is mixed by counter-current Taylor vortices. A continuous-wave or a pulsed laser-UV source is disclosed for illumination.
Similar Taylor-vortex generating devices has also been investigated as photochemical reactors for heterogeneous and homogeneous photochemical processes (Sczechowski et al. 1995, Forney and Pierson 2003). For the actinometric examination of the reactor, either the ferroixalate (Kirk and Mamasivayam 1983) or the iodide/iodate actinometer (Rahn 1997) were used. The iodide-iodate actinometer was also diluted up to 100-fold to reduce the iodide concentration for the examination of the relationship of the outlet triiodide concentration to the inlet iodide concentration (Forney and Pierson 2003). The relationship of the outlet triiodide concentration to the flow-rate dependent 254 nm photon dose was determined using a fixed absorbance of the actinometer solution, and a theoretical calculation based on the lamp geometry, the flow-rate, the quantum yield and the photon energy. However, an absorbance-matching calibration to determine the dose effective in the iodide solution was neither proposed nor done ((Forney and Pierson 2003).
WO 97/33629 discloses a process for sterilization and purification of biological fluids by exposure to UV radiation between 200 nm and 250 nm.
In 1960, one of the leading experts in thin-film UV-C-irradiated vaccine technology stated: “In order to calculate the absolute quantity of energy involved in the virus inactivation itself, there must be some manner of quantitating the relative amounts of ultraviolet energy absorbed by the virus and the culture medium. This has not been possible as yet; furthermore the absolute exposure is dependent upon a number of variable factors: viscosity, temperature, surface tension, and frictional resistance of flow” (Taylor 1960). Prior to the present invention, the scientific or patent literature still fails to disclose a determination of the photochemically effective dose on pathogens in a fluid sample. Accordingly, a method for the inactivation of pathogens in a biological fluid is desirable. A method for determining a photochemically effective dose of electromagnetic radiation, such as light, sufficient to inactivate pathogens in a biological sample while leaving biologically active substances of interest unaffected is also particularly desirable.